Mammary Tissue Slices Research Articles

Roles of Lactate and Its Interactions with Acetate in Maintenance and Biosynthesis in Bovine Mammary Tissue

Mammary gland tissue slices from three lactating Holstein cows were incubated in Krebs-Ringer bicarbonate-based media with varying concentrations of lactate and other substrates. Conversions of 1- and 2-carbon-14 of lactate to carbon dioxide, fatty acids, citrate, glyceride glycerol, and lactose were determined.Effects of acetate, glucose, beta-hydroxybutyrate, stearate, and pyruvate on lactate metabolism were evaluated. Oxidation of lactate increased asymptotically with lactate concentration. Low acetate concentration stimulated oxidation of carbon 2 of lactate slightly while higher acetate availability inhibited lactate oxidation. Conversion of lactate to fatty acids increased linearly with lactate concentration. This conversion of lactate was inhibited strongly by acetate. Significant conversion of carbon 2 of lactate to glyceride-glycerol but not lactose was detected.Bovine mammary glands have the capacity of utilize sizeable quantities of lactate for oxidation and lipogenesis. Both phenomena are highly dependent on availability of acetate. Pyruvate dehydrogenase and citrate lyase could represent important regulatory sites in vivo for determination of tissue preference for acetate over lactate. Results indicate factors other than an inactive malate transhydrogenation cycle likely limit glucose conversion to fatty acids.

A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle

The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a β-adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle.

Effects of Propionate and Methylmalonate on Conversions of Acetate, Butyrate, and D(−)-3-Hydroxybutyrate to Fatty Acids and Carbon Dioxide by Mammary Tissue Slices of Goats

Incorporations of [1-carbon-14] acetate, [1-carbon-14] propionate, n-[1-carbon-14] butyrate, and D(−)-3-hydroxy [3-carbon-14] butyrate into individual milk fatty acids and their conversion to carbon dioxide were studied in vitro with caprine mammary tissue slices in the presence and absence of propionate and methyl-malonate. Neither propionate nor methyl-malonate affected incorporation of these substances into fatty acids. In a decreasing order butyrate, acetate, propionate, and D(−)-3-hydroxybutyrate were converted to carbon dioxide. Acetate had the highest incorporation rate into fatty acids followed by D(−)-3-hydroxybutyrate, butyrate, and propionate. Labeled propionate was incorporated mainly into odd-numbered fatty acids. Results do not support the theory that either propionate or its metabolite, methylmalonate, inhibit de novo synthesis of fatty acids in the mammary gland in relation to the etiology of low milk fat syndrome.

Effect of prepartum blockade of microtubule formation on milk production and biochemical differentiation of the mammary epithelium in holstein heifers

1. 1. Prepartum intramammary infusions of colchicine markedly reduced subsequent milk production compared with untreated mammary glands in the same animal. 2. 2. After the first week postpartum, milk composition was similar in control and treated mammary glands. 3. 3. Rates of fatty acid synthesis, CO 2 production and protein synthesis at either 5 or 21 days postpartum were lower in mammary tissue slices taken from mammary glands treated prepartum with colchicine. 4. 4. We conclude that an intact microtubule system is necessary for initiation of milk synthesis and secretion at parturition.

Effects of hormones on mammary adenosine 3',5'-monophosphate levels and metabolism in normal and adrenalectomized lactating rats.

Prostaglandin E1 (PGE1) T4, and epinephrine (Epi) were incubated with mammary tissue slices from normal and adrenalectomized (Adx) rats and Adx rats receiving cortisol (Adx + C). PGE1, T4, and the combination of Epi, T4, and PGE1 increased mammary slice cAMP levels in all groups. No differences were observed among treatment groups in basal cAMP levels or responses to hormones. Glucose-1-14C oxidation and incorporation into lipids were stimulated by PGE1, T4, and the combination of EPi, T4, and PGE1 in normal rats. Responses were depressed by adrenalectomy and reversed by cortisol. PGE1, T4, and the combination of EPi, T4, and PGE1 depressed glucose-6-14C oxidation. Fatty acid synthesis from glucose-6-14C was stimulated by Epi, PGE1, and T4 in tissues from normal and Adx + C rats, but not in those from Adx rats. Glycerol synthesis was unchanged. Pyruvate-1-14C and pyruvate-2-14C oxidation were uneffected by adrenalectomy or hormones. Fatty acid synthesis was depressed by adrenalectomy and reversed by cortisol. T4 and the combination of Epi, T4, and PGE1 depressed triacylglycerol and fatty acid synthesis in all groups. Glycerol synthesis was unchanged. Hormonal effects were much more pronounced when glucose was the substrate. This suggests that cAMP effects on glucose metabolism are manifest at a site between hexose-P and pyruvate, and further, that a primary effect of adrenalectomy may be to reduce the responsiveness of this site to cAMP. Linear regression analyses comparing cAMP levels with glucose and pyruvate metabolic parameters further support this conclusion.

Methylmalonic Acid in Low-Fat Milk Syndrome

The possible association between low-fat milk syndrome and methylmalonic acid accumulation in blood was investigated. Blood was sampled from the internal iliac artery of nine lactating dairy cows fed ad libitum roughage plus grain or high grain, restricted roughage diets. Daily milk fat percent and milk fat production were decreased 44% and 47% on the high grain, restricted roughage diet. No differences in blood methylmalonate concentrations could be detected in the cows fed the two diets. The effect of methylmalonate and its metabolic precursor, propionate, on mammary rates of fatty acid synthesis was determined with bovine mammary tissue slices. Neither of these metabolites affected acetate incorportation into fatty acids. Methylmalonate and propionate were incorporated into fatty acids at extremely low rates. Low-fat milk syndrome is not caused by accumulation of methylmalonic acid.

Palmitate and Octanoate Metabolism in Bovine Mammary Tissue

Effect of glucose on palmitate and octanoate metabolism was studied in bovine mammary tissue slices. Glucose stimulated palmitate esterification principally to form partial glycerides while decreasing palmitate oxidation. Glucose stimulated octanoate but not palmitate incorporation into triacylglycerols, suggesting that availability of intracellular medium-chain fatty acids may limit the final acylation reaction in triacylglycerol synthesis.

Factors affecting fatty acid oxidation in bovine mammary tissue.

AbstractOxidation of fatty acids was studied in bovine mammary tissue slices in order to evaluate their potential contribution to energy metabolism. Rates of fatty acid oxidation decreased with increasing chain length: acetate>octanoate>palmitate or oleate. Rates of oxidation of long chain, but not short chain, fatty acids increased over time, which could not be explained by carnitine palmitoyltransferase (CPT) activity. This phenomenon is not an artifact of the incubation system or caused by substrate solubility, as rates of palmitate oxidation were constant in rat kidney cortex slices. Preincubating mammary tissue with or without unlabeled palmitate showed that increasing rates of palmitate oxidation is not caused by use of endogenous fatty acids. Palmitate at 0.26 mM, equivalent to arterial fatty acid concentration, gave maximal rates of oxidation. The β‐oxidation enzymes may restrict fatty acid oxidation as oxidation of [1‐14C] palmitate exceeded that of [U‐14C] palmitate. Acetate inhibited palmitate oxidation (75%) but not esterification, suggesting that acetate inhibits palmitate oxidation by substrate competition at the mitochondrial level or via malonyl‐CoA inhibition of CPT. Glucose inhibited palmitate oxidation (67%) and stimulated esterification. Low palmitoyl‐CoA levels would favor glyceride synthesis over oxidation, since the apparent Km for palmitoyl‐CoA, of the glycerol‐3‐phosphate acyltransferases is lower than that for CPT. Thus, glucose presumably diverts palmitate from oxidation to glycerolipids. Clofenapate, a glyceride synthesis inhibitor, decreased triacylglycerol formation, and marginally increased palmitate oxidation. We estimated that long chain fatty acids can potentially account for 6–10% of the oxidative metabolism of mammary tissue.

Progesterone inhibition of glucocorticoid binding to mammary tissue from lactating and nonlactating cows.

AbstractMammary tissue cytosols (105,000g supernatants) were prepared from lactating or from nonlactating, nonpregnant cows, then incubated with [3H]dexamethasone alone (3 × 10-8M) or in combination with various unlabeled hormones (1.9 × 10-7 or 1.9 × 10-6M). Cortisol, corticosterone, and progesterone inhibited binding of [3H]dexamethasone, whereas testosterone, cortisone, and estradiol-17 β did not. Binding affinities of mammary cytosols from lactating and nonlactating cows for [3H]dexamethasone (0.5 to 100 nM) were virtually identical averaging 1.4 (± 0.3) and 2.0 (± 1.2) × 10-9M, respectively. However, binding capacities of mammary tissue slices (fmole dexamethasone/μg DNA) were greater in lactating (2.3) than in nonlactating (1.5) cows. Specific cytosolic binding sites sedimented at 8 and 4 S on sucrose gradients. Whole mammary slices were incubated with 3 × 10-9M [3H]dexamethasone alone or in combination with 10-13 to 10-5 × 3.8 M progesterone or 3 × 10-7M dexamethasone, and cytoplasmic and nuclear f...

Inhibition of cholesterol synthesis in mammary tissue, lung, and kidney following cholesterol feeding in the lactating rat.

Pregnant rats were randomly allocated to one of 3 experimental dietary groups: Group 1 -- 15.5% butter, 2% cholesterol, 0.78% sodium cholate purified diet; Group 2 -- standard rat diet with the addition of 10% lard and 2% cholesterol, and Group 3 -- standard rat diet. Plasma and milk cholesterol at 10 days postpartum were significantly elevated in dams fed exogenous cholesterol. The rat of incorporation of [1-14C] acetate into digitonin-precipitable sterols of mammary tissue slices from dams in Group 1 and Group 2 was eight-fold and two-fold, respectively, less than controls. Mammary tissue cholesterol was greater in dams fed detary cholesterol. Thus, our data, for the first time, demonstrate that cholesterol synthesis in lactating rat mammary tissue is suppressed following cholesterol feeding. In a second experiment, the rate of incorporation of [1-14C] acetate into digitonin-precipitable sterols in kidney and lung tissue of Group 1 rats was suppressed; however, this response was not as marked as that observed in lactating mammary tissue. The concentration of cholesterol in kidney and lung was greater than controls. These results suggest that extrahepatic inhibition of cholesterol synthesis exists in the rat with a concomitant increase in tissue cholesterol.

Valine, Leucine, and Isoleucine Metabolism by Lactating Bovine Mammary Tissue

Valine, leucine, and isoleucine were extracted by the lactating bovine mammary gland in excess of outputs in milk protein. Carbon-14 uniformly labeled L-valine, L-leucine or L-isoleucine were catabolized when incubated in vitro with lactating bovine mammary tissue slices. The pathways of degradation of these amino acids by mammary tissue appear to be those common to other tissues. These amino acids represent a potential source of energy to the mammary gland as well as a source of carbon and α-amino nitrogen for synthesis of nonessential amino acids.

Lactational Events Related to Glucocorticoid Binding in Bovine Mammary Tissue

Various synthetic glucocorticoids reduced binding of tritiated Cortisol and tritiated dexamethasone to 700×g supernatant and precipitate fractions of mammary tissue slices from lactating cows. Unlabeled progesterone, testosterone, and 17β-estradiol had no effect on tritiated glucocorticoid binding in mammary tissue slices. Cortexelone, Cortisol, triamcinolone, and dexamethasone inhibited [carbon-14] glucose incorporation into mammary tissue slices from lactating cows in a dose response relationship. Glucocorticoid binding to 700×g supernatant and precipitate fractions of mammary tissue was correlated with the ability of glucocorticoids to reduce [carbon-14] glucose uptake into 700×g supernatant fractions of mammary tissue slices from lactating cows. In experiments designed to measure mammary uptake of glucocorticoids, the differences in concentration of total glucocorticoids between external pudic artery and mammary vein were greatest at 6 (5.8ng/ml) and 12 (2.9ng/ml) min after the start of milking. These intervals correspond to the times when glucocorticoid concentrations in serum were maximal after application of the milking stimulus. We conclude that glucocorticoid uptake and binding are associated with lactational events.

Glucocorticoid Binding in Mammary Tissue Slices of Cattle in Various Reproductive States

Unlabeled cortisol and dexamethasone reduced tritiated cortisol and tritiated dexamethasone binding to 700 X g supernatant and precipitate fractions of mammary tissue slices from virgin heifers and from multiparous cows that were 1-mo prepartum (nonlactating), lactating (non-pregnant), or dry (nonpregnant, nonlactating). Unlabeled progesterone, testosterone, and 17beta-estradiol had no effect on tritiated glucocorticoid binding in 700 X g supernatant and precipitate fractions from these mammary tissue slices. The 700 X g fractions in mammary tissue slices from all cattle bound cortisol and dexamethasone with high affinity (Kd 10-10M). There were 1263 and 1955 molecules of cortisol and dexamethasone bound per mammary cell, respectively, in mammary tissue slices from lactating non-pregnant cows; in comparison virgin heifers bound 413 and 651 molecules of cortisol and dexamethasone; dry, non-pregnant cows bound 336 and 536 molecules of cortisol and dexamethasone; and 1-mo prepartum nonlactating cows bound 532 molecules of cortisol. Mammary tissue slices from cattle in reproductive states examined contained a major non-specific component which bound cortisol in both 700 X g tissue fractions. Since mammary tissue slices from lactating cattle bound more molecules of glucocorticoids than mammary slices from cattle in other reproductive states, we speculate specific glucorticoid binding may be associated with lactation.

Comparison of Binding Proteins of Glucocorticoids in Mammary Tissue and in Blood Sera from Lactating Cows

Supernatant fractions (700 X g) isolated from homogenates of mammary tissue slices from three lactating Holstein cows (slices previously incubated with [hydrogen-3] cortisol for 1 h at 37 C) were electrophoresed on 7% polyacrylamide gels. The majority of radioactivity was in a protein(s) 2.5 to 3 cm from the origin whereas bovine serum incubated with [hydrogen-3] cortisol showed the majority of radioactivity in a protein 5 to 6 cm from the origin. N,N-diethylaminoethyl cellulose chromatography of 700 X g supernatant fluids from three lactating cows revealed that [hydrogen-3] cortisol was associated with a protein component that eluted with .3 M potassium phosphate, pH 8.0. In contrast, [hydrogen-3] cortisol bound to bovine sera eluted as two protein components with .05 and .1 M potassium phosphate, pH 8.0. Approximate molecular weights determined from gel filtration (four lactating cows) and sucrose density studies (two lactating cows) were estimated to be 2.5 X 10(5) to 3 X 10(6) for the 700 X g mammary receptor of cortisol and 6 X 10)4) to 8 X 10(4) for the primary protein which binds cortisol in serum. We conclude that lactating bovine mammary tissue contains a protein(s) capable of binding tritiated cortisol which is unique from the corticosteroid binding proteins of blood.

Effects of Adrenalectomy upon Ruminant Liver and Mammary Function during Lactation

Adrenalectomized, lactating ewes treated with a mineral corticoid or a mineral corticoid plus cortisol and sham operated ewes nursing one or two lambs were in a study of physiological role(s) of glucocorticoids in lactating ruminants. Measurements of ewe and lamb weight changes; feed intake; milk yield and composition; blood potassium; metabolite oxidation rates by mammary, liver, and kidney tissue slices; glucose production in liver and kidney tissue slices; and enzyme activities in mammary and liver tissue. 1) Mammary metabolism and milk biosynthesis in ruminants are not strongly dependent on glucocorticoid. 2) Liver gluconeogenesis in ruminants appears to be regulated in part by glucocorticoid(s) but less prominently than in nonruminants. 3) Liver and kidney of ruminants may respond differently to adrenalectomy; thus, in ruminants, kidney may not be as good an indicator of liver gluconeogenic responses as it is in rodents. 4) Liver and mammary enzymes are not affected significantly by adrenalectomy or glucocorticoid therapy. 5) Glucocorticoids play a less significant role in the regulation of liver and mammary enzyme and metabolism in sheep than in rats.

Corticoid Binding in Mammary Tissue Slices from Lactating Cows

Mammary tissue slices (from lactating Holstein cows) incubated at 37C with various concentrations of either hydrogen-3 cortisol or hydrogen-3 dexamethasone bound both hormones with high affinity [dissociation constants (Kd)≅10−10M]. There were approximately 2900 and 3800 total high affinity binding sites per mammary cell for cortisol and dexamethasone, respectively. In addition, a major nonspecific component bound cortisol and dexamethasone and was unsaturable. Unlabeled cortisol and dexamethasone reduced binding of hydrogen-3 cortisol and dexamethasone whereas unlabeled 17β-estradiol, testosterone, and progesterone were without effect. Incubation of tissue slices at 4C reduced the total number of high affinity binding sites for cortisol by about 56% compared with similar measurements at 37C. However, dissociation constants for the high affinity components were similar at both temperatures (Kd≅10−10M). Macromolecules which specifically bound cortisol in the 700×g supernatant and precipitate tissue fractions were isolated by gel filtration chromatography. Enzyme digestion experiments and treatment with p-chloromercuribenzoate indicated that the macromolecules binding cortisol were proteins. Thin-layer chromatography of bound hydrogen-3 cortisol in the 700×g supernatant indicated that the majority of bound radioactivity was authentic cortisol. We conclude that fresh mammary tissue from lactating cows possesses protein which specifically binds corticoids with high affinity

Cellular Gluconeogenesis by Lactating Bovine Mammary Tissue

The gluconeogenic capacity of mammary tissue of lactating cow was investigated by incubating mammary tissue slices with alanine, glutamate, lactate, pyruvate, or glycerol in conjunction with acetate and glucose (10mM or 1mM). In no case was any substrate incorporated into glucose per se. In lactose synthesis, glucose was the mjaor source of carbon although glycerol also was incorporated into lactose. Alanine, glutamate, lactate, or pyruvate were not incorporated into lactose at optimum (10mM) or suboptimum (1mM) concentrations of glucose. Activity of glucose-6-phosphatase was negligible in mammary tissue, less than 1% of the activity in liver or kidney tissue from the same cows. Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and fructose-1,6-diphosphatase were in cow mammary tissue, but the activities were lower than in liver. Gluconeogenic substrates were not converted to glucose regardless of whether the incubation contained an optimum (10mM) or a suboptimum (1mM) glucose concentration. Consistent with the inability of cow mammary tissue to convert gluconeogenic metabolites to glucose is the virtual absence of glucose-6-phosphatase and the lack of excess gluconeogenic substrates available to the intact mammary gland of lactating cow.

Stimulation of DNA synthesis by prolactin in rabbit mammary tissue.

ABSTRACTThe capacity of mammary gland tissue slices to incorporate [3H]thymidine into DNA was studied at intervals after intraductal injection of prolactin. Prolactin markedly increased the rate of incorporation of thymidine into DNA (as extracted by HClO4 or by sodium dodecyl sulphate). The increase in incorporation rate occurred within 6 h of prolactin injection. Autoradiography of mammary tissue, after administration of [3H]thymidine in vivo, showed that the increased rate of incorporation of thymidine was caused by an increase in the number of alveolar cells entering 5-phase. These results are discussed in relation to current theories concerning functional differentiation of mammary tissue.

Effects of prolactin on the synthesis of casein in rabbit mammary tissue

1. 1. The ability of mammary gland tissue slices to incorporate 32P-orthophosphate into casein was studied at intervals after intraductal injection of prolactin. 2. 2. The rate of incorporation increased during the first 24 hr after prolactin injection, and then declined over the next 24 to 48 hr. 3. 3. The results obtained by polyacrylamide gel electrophoresis suggested that the increase in incorporation rate during the 24 hr following prolactin injection was accompanied by a change in the pattern of incorporation into individual casein components.

Factors Affecting In Vitro Lipogenesis by Bovine Mammary Tissue Slices

An in vitro system of fatty acid synthesis by bovine mammary tissue slices was developed and characterized. Mamary tissue slices were prepared wih a hand microtome and 120 to 150-mg slices incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) at 37C. Acetate incorporation into fatty acids and acetate and glucose oxidation to carbon dioxide were linear for 3h. Mammary tissue was stable and lost no lipogenic capacity for 60min prior to slicing and incubation if maintained in isotonic sucrose at 5 or 25C. Fatty acid synthesis from acetate was maximum at an acetate concentration of 10mM and a glucose concentration of 5 to l0mM. Insulin had no effect on acetate incorporation into fatty acids by cow mammary tissue slices when varied from physiological to pharmacological concentrations. An examination of lipids synthesized by bovine mammary slices indicated that over 70% of the synthesized fatty acids were located in triglycerides with the pattern of fatty acids similar to that produced in vivo. The use of the in vitro system of bovine mammary tissue slice incubations offer an excellent tool for investigation of cellular metabolism and biosynthesis of fatty acids.